Calmodulin purified from human and porcine thyroids inhibits thyrotropin binding to porcine thyroid cells.
A thyrotropin (TSH) binding inhibiting protein (TBIP) that inhibits TSH binding to the TSH receptor, as determined by the TSH receptor assay, was purified from human and porcine thyroid. The soluble fraction (100,000 x g supernatant of Graves' thyroid homogenate) was precipitated with ammonium sulfate between 1.75 to 2.5 mol/L. TBIP was eluted by 0.5 mol/L sodium chloride (NaCl) containing 20 mmol/L Tris buffer, pH 7.5 from a Q-sepharose column. The unbound fraction from concanavalin A (Con A) and blue-sepharose was gel-filtered using sephadex G-100, and finally purified by Resource Q column chromatography. Purified TBIP was confirmed as a single protein band of 17 kDa. The TBI activity in the purified TBIP was significantly decreased by either etnylene glycol tetraacetate (EGTA) (1 mmol/L) or antibody to calmodulin (CaM) in the TSH receptor assay. The TBIP was confirmed immunologically as CaM by the Ouchterlony method using antibody for CaM. These findings demonstrated that the TBIP purified from human and porcine thyroids was, in fact, CaM. We examined the effects of TBIP purified from human thyroid on bovine TSH (bTSH) or thyroid stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cells (PTC). TBIP itself did not increase basal levels of cAMP production, but inhibited bTSH (100 mU/L)-stimulated cAMP production. However, TBIP did not inhibit cAMP production stimulated by TSAb-IgG and various thyroid stimulators (GTPgammaS, forskolin and pituitary adenylate cyclase-activating polypeptide [PACAP, 27 and 38 amino acids]). Authentic CaM purified from bovine brain behaved in a manner similar to that of TBIP. These data showed that CaM differentially affects thyroid stimulation by TSH and TSAb in intact thyroid cell experiments.
Kouki
T
T
Central Clinical Laboratory, Shiga University of Medical Science, Otsu, Japan.
Inui
T
T
Hachiya
T
T
Ochi
Y
Y
Kajita
Y
Y
Sato
Y
Y
Nagata
A
A
Ozaki
O
O
Ito
K
K
Kurihara
H
H
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002147:Calmodulin / Q000187:drug effects / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D003208:Concanavalin A; D000242:Cyclic AMP / Q000378:metabolism; D003911:Dextrans; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006111:Graves Disease / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007457:Iodine Radioisotopes; D011485:Protein Binding / Q000187:drug effects; D011989:Receptors, Thyrotropin / Q000187:drug effects / Q000378:metabolism; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D013961:Thyroid Gland / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects*; D013972:Thyrotropin / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D000906:Antibodies; D002147:Calmodulin; D003911:Dextrans; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D003208:Concanavalin A; D012967:Sodium Dodecyl Sulfate; D004533:Egtazic Acid; D013972:Thyrotropin; D012685:Sepharose; C025614:sephadex; D000242:Cyclic AMP